Ünüvar S. JOTCSA. 2018; 5(3): 1431-1440. RESEARCH ARTICLE
Determination of 5-hydroxymethylfurfural (5-HMF) in Expired Pharmaceutical Syrups by Using HPLC-DAD Method
Songül Ünüvar*
University of Inonu, Faculty of Pharmacy, Department of Pharmaceutical Toxicology, 44280,Malatya, Turkey.
Abstract: The Maillard reaction product 5-hydroxymethylfurfural (5-HMF) is formed under acidic conditions by the dehydration of sugars in carbohydrate-based food and pharmaceutical products during heating and storage. As pharmaceutical syrup formulations contain sugar and are stored under room temperature, they provide favorable conditions for the formation of 5-HMF. The long-term storage of syrup bottles after their cap has been opened and the unintentional use of expired syrups can lead to the formation of undesirable products such as 5-HMF in medications. Although legal limits have been established for 5-HMF content in pharmaceutical preparations, these levels may exceed those limits in hot climates or under inappropriate storage conditions. The present study detects and measures 5-HMF levels in expired pharmaceutical syrups through the HPLC-DAD (High Performance Liquid Chromatography with Diode Array Detection) method, and investigates the effects on 5-HMF levels of the 72-hour storage of syrups at temperatures of 40 °C. The 5-HMF level in syrups stored at room temperature varied between 1.34 μg/mL to 15.63 μg/mL, while in syrups stored at higher temperatures, the levels ranged from 2.24 μg/mL to 18.24 μg/mL. This indicated that 5-HMF content in syrups stored at 40 °C was higher than those measured in syrups stored at room temperature, although the increase was not found to be statistically significant (p>0.05). In addition to measuring the amount of 5-HMF in pharmaceutical syrups, this study also examined the changes in the levels of this dehydration product in syrup formulations under hot climates and according to storage conditions.
Keywords: 5-hydroxymethylfurfural (5-HMF); pharmaceutical syrup; HPLC-DAD; exposure; sucrose; expiration date.
Submitted: November 02, 2018. Accepted: December 28, 2018.
Cite this: Ünüvar S. Determination of 5-hydroxymethylfurfural (5-HMF) in Expired Pharmaceutical Syrups by Using HPLC-DAD Method. JOTCSA. 2018;5(3):1431-40.
DOI: http://dx.doi.org/10.18596/jotcsa.477710.
*Corresponding author. E-mail: [email protected]. Tel: +90 506 245 40 54, Fax: +90 422 341 12 17.
INTRODUCTION reaction (6). Upon heating food that contains sugar or carbohydrates, it forms as a result of The compound 5-HMF consists of a furan ring hexose reduction reaction in the presence of along with aldehyde and alcohol functional amino-acids or proteins (7). groups. Its chemical formula is 5- (hydroxymethyl)-2-furancarboxaldehyde The presence of 5-HMF has been reported in (C6H6O3) (1), and it is used in the synthesis of several foods, including honey, grain products, certain organic compounds (2) and novolac resins biscuits, cereals, UHT milk, tomato products, (3). It is also used as an intermediate substance instant coffee, dried fruits, bread, pasta, citrus in the synthesis of some crown ethers (4), and in juices, beer, syrup, jams, canned peach, dried the production processes of some polymers, grape, alcohol, apple juice, milk and cereal-based surfactants, solvents, pharmaceutics and plant infant formula. The presence of 5-HMF in foods protection agents (5). It is one of the most reflects a breakdown or change of substances significant products of non-enzymatic Maillard containing sugar, which is why 5-HMF levels in
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Ünüvar S. JOTCSA. 2018; 5(3): 1431-1440. RESEARCH ARTICLE food are generally analyzed for quality control without prior pretreatment (25). The real time purposes (8-12). Food processing conditions, coupled with time of flight mass spectrometry is such as temperature, time and water activity, another method used in 5-HMF analysis (26). affect 5-HMF content in foods. The daily uptake of Based on the information from the literature 5-HMF in foods may occasionally reach 150 review, we conclude that the differences between mg/day (13) and it may be present in foods at the methods cause very low levels of changes in varying levels (10,14). Very high levels of 5-HMF the 5-HMF results. On the other hand, the use of can be found in such foods as dried fruits or incorrect or inadequate procedures in the 5-HMF caramelized products (> 1 g/kg) (7). In addition determination leads to inaccurate results. We to caramelized foods, 5-HMF has also been preferred the HPLC-DAD method among these identified in caramel-colored pharmaceutical listed methods. Because this method is a rapid, syrups (15). Although the concentrations sensitive and automated method that separates reported in pharmaceutical syrups are very low, 5-HMF from other related compounds in syrup there are concerns about the potential samples and prevent interference in the interactions between 5-HMF and functional amino determination. To our knowledge this is the first groups of pharmaceutics (11). study to measure 5-HMF amount in pharmaceutical syrups by using HPLC-DAD The formation of 5-HMF occurs as a result of the method. Apart from syrups, several other acid-catalyzed dehydration process of fructose, pharmaceutical preparations, including tablets, sucrose and, to a lesser extent, glucose. As a capsules, micropellets, pills, ampules, result, it may, in addition to food, also be found mouthwashes, pomades and creams, tend to be in heat-sterilized parenteral nutritional solutions stored at room temperature, which is defined as containing glucose/fructose (16). The 25 °C degrees or lower (15–25 °C). However, in quantitative analysis of 5-HMF in clinical research Turkey, ambient temperatures may exceed 40 °C and therapeutics is of great importance as in during the summer. It is known that 5-HMF levels foods (17). Various methods have been defined increase in foods and pharmaceutical for the measurement of 5-HMF levels, including preparations stored at high temperatures and for colorimetric, spectroscopic, chromatographic, long durations after production. polarographic and two spectrophotometric methods; White’s method and Winkler’s method The present study investigates the effects of post- (6,18,19). HPLC method and spectrophotometric expiration temperatures on 5-HMF levels in methods were recently tested by the pharmaceutical syrup formulations exposed to International Honey Commission (IHC) (20). The high temperatures after their expiration date. first used before the spectrophotometric methods were optical and chemical methods (17). The MATERIALS AND METHODS basis of the White’s method is based on the measurement of UV absorbance of clarified Chemicals aqueous honey solutions with and without The 5-Hydroxymethyl-2-furfural (5-HMF) was bisulfite. In the other spectrophotometric Winkler obtained from Dr. Ehrenstorfer GmbH method, the UV absorbance of honey solutions (Germany). Methanol was analytical grade and with barbituric acid and p-toluidine is measured. was obtained from Merck (Darmstadt, Germany). Although these two methods are fast, their Ultrapure water was used in all experiments sensitivity and specificity are not sufficient. In (Milli-Q system, Millipore, Bedford, MA). addition, the use of carcinogen p-toluidine in the Winkler’s method is a disadvantage. The Preparation of stock and standard solutions disadvantage of the HPLC method is that it is On the day of the experiment, 10 mg of high more expensive, but it provides advantages in purity (>98%) 5-HMF standard was weighed terms of both labor and time (20,21). In the HPLC using an analytic scale and completed to 100 mL method is according to Jeuring and Kuppers: with ultrapure water in a 100 mL volumetric flask. firstly honey is dissolved in water. 5-HMF is This provided 100 ppm of stock standard solution. determined on a reversed phase HPLC column From this stock standard, 1, 2, 5, 10 and 50 mL with water and methanol as isocratic mobile solutions were transferred to 100 mL volumetric phase after millipore filtration (21). Borate is used flasks and the volumes were completed to the as supporting electrolyte in electrochemical mark line using ultrapure water, which provided method. The basis of the method is a single and 1, 2, 5, 10 and 50 ppm standard solutions. sharp reduction signal against the silver or silver chloride (22). Yuan et al. have used the ion Preparation of samples exchange liquid chromatography with photodiode The study was carried out using 10 different array detection technique (23). Another method pharmaceutical syrup samples obtained from the used in 5-HMF analysis is the automated flow pharmacy. The syrups were kept at room injection method which provides a detection temperature (25±2 °C) for 72 hours. Separate 30 range of 5-40 ppm (24). Caffeine is used as an g samples were taken from each syrup and stored internal standard in micellar electrokinetic for 72 hours in an incubator set to 40±2 °C capillary chromatography, which is used in 5-HMF (Thermo Scientific Heratherm). Then, 10 grams analysis. This technique allows rapid of the samples were taken from the syrups kept quantification of the sample, especially in honey at both temperatures and dissolved in 25 mL of 1432
Ünüvar S. JOTCSA. 2018; 5(3): 1431-1440. RESEARCH ARTICLE ultrapure water, quantitatively transferred to a 50 20 µL. Monitoring of the analytes was carried out mL volumetric flask and completed to 50 mL with using a DAD detector at 284 nm wavelength (27). ultrapure water. The flasks were placed in a shaker for 15 minutes to ensure complete The determined limit of detection (LOD, S/N=3) dissolution of the syrup. Before injection into the and limit of quantification (LOQ, S/N=10) values HPLC column, the solutions were sampled into for 5-HMF substances were 0.011 μg/mL and syringes and passed through a 0.45 µm filter 0.036 μg/mL, respectively. The linearity of the (SIMPLEPURE) and then transferred to 2 mL method used was tested in the concentration amber vials. The 5-HMF samples were kept range of 1-50 mg/L by means of an 5-HMF protected from light and air throughout the study. standard (GmbH, Germany).
Instruments Statistical analysis Chromatography analyses were carried out with The statistical analysis of the data was carried out an Agilent 1100 HPLC device, which comprises a using the SPSS Version 11.5 statistical package degasser (G1379A), quaternary pump (G1311A), software (SPSS Inc., Chicago, IL, USA) and was autosampler (G1313A) and diode array detector expressed as mean±SD. A Mann-Whitney U-test (DAD) model G1315. Separations were carried was used for the comparison of the two out in an ACE C18 column, 250 x 4.6 mm x 5 µm independent groups and a Pearson correlation particle sized. The mobile phase used was test was used in the evaluation of the methanol: water (90: 10, v/v); the prepared correlations. P values <0.05 were considered mobile phase was placed into the HPLC device statistically significant. and passed through the column at a flow rate of 1 mL/min to condition the column. The samples RESULTS AND DISCUSSION transferred to the vials were injected into the HPLC system. Flow rate: 1 mL/min. Injection The analyzed syrups contained raspberry, time: 20 min. The temperature of the column orange, grape and mixed-fruit sweeteners, and compartment was 25 °C and injection volume was all of the samples had reached their expiration date (Table 1).
Table 1: Flavor and sweetener contents of the expired pharmaceutical syrups. Sample name Expiration date (month) Sweetener Flavor S1 2 Sucrose - S2 4 Sucrose Mixed-fruits S3 15 Glycerin Mixed-fruits S4 11 Glycerin Orange S5 12 Sucralose Mixed-fruits S6 5 Sucrose Raspberry S7 6 Sodium saccharine Mixed-fruits S8 4 Sucrose Orange S9 15 Sodium saccharine Raspberry S10 1 Sucrose Grape
Figure 1 shows the calibration curve of standard peak level for 5-HMF was attained at minute solutions, and the chromatograms of standard 10.493 (Figure 1C). 5-HMF was not detected in solutions of 5-HMF and a syrup sample injected not expired syrup sample of S7 (Figure 1D).Three with 5-HMF content. The calibration curve was replicates of syrup samples were analyzed. The drawn using peak areas of increasing mean 5-HMF level in the syrup samples stored at concentrations of standard solutions (1, 2, 5, 10, room temperature was 7.50 μg/mL, while the and 50 μg/mL) (Figure 1A). Calibration curve of mean 5-HMF level in the incubated syrup samples increasing concentrations of 5-HMF standards was 8.88 μg/mL. A total of four samples were was drawn by evaluating five replicates of each. studied from each syrup sample to keep two of them in room temperature and the other two in Retention time was estimated as minute 10.038 incubation. Table 2 shows the 5-HMF levels of for the chromatogram of the 10 μg/mL standard syrups stored at different temperatures for 72 solution (Figure 1B). In the syrup sample, the hours.
Table 2: 5-HMF concentrations in syrups stored at different temperatures (μg/mL). Sample name 25±2 ºC 40±2 ºC S1 4.94±0.11 6.54±0.80 S2 6.98±0.14 9.10±0.18 S3 n.d. n.d. S4 n.d. n.d. S5 1.34±0.31 4.85±1.24 S6 2.87±0.03 2.24±0.20 S7 15.63±0.92 18.24±2.08 S8 10.84±1.37 11.56±1.22 S9 13.21±0.20 13.92±1.33 S10 4.20±0.42 4.59±0.41 n.d: not detected. Results are presented as mean ± SD. 1433
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Figure 1. A) Calibration curve of standard solutions of 5-HMF. B) Chromatogram of injected standard 5- HMF C) Chromatogram of 5-HMF in a expired syrup sample.D)HPLC result of not expired pharmaceutical syrup sample.
Although heat treatment increased 5-HMF different expiration dates (Figure 2). Although a content in all samples except one, the increases positive correlation was found, the relationship were not statistically significant (p: 0.638). between the variables was weak (r: 0.227) and insignificant (p: 0.588). The relationship between time after expiration and 5-HMF content was evaluated for syrups with
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Figure 2. Correlation between time after expiration and 5-HMF content.
Human exposure to 5-HMF essentially occurs Later studies have suggested that this is not the through the consumption of processed foods and case, and the carcinogenic activity of 5-HMF has beverages, the use of pharmaceutical been rejected through direct or indirect preparations and the inhalation of tobacco smoke. investigations (34). Apart from the absence of It may also occur occasionally through established genotoxic, mutagenic or carcinogenic occupational exposure as a result of the inhalation activities, some studies in recent years have of the vapor of solutions including 5-HMF, and reported that 5-HMF may even have some dermal contact with these compounds in facilities favorable effects, for example, as a potential new producing or using 5-HMF-derived polymers or antioxidant in the fight against cancer (35). It is chemicals (11). also shown to have beneficial physiological effects, such as reducing oxidative stress 5-HMF is metabolically activated through the resulting from high glucose levels (36), sulfonation of the allylic hydroxyl functional neuroprotective effects (37), anti-hypoxic effects groups by sulfotransferases (SULT1A1), and its (38), anti-allergenic effects (39) and anti- bioactivation results in the formation of 5- inflammatory activity (40). sulfoxymethylfurfural (SMF) (28,29). It shows no effect in standard genotoxicity tests, but its While several countries have established limits for mutagenic and carcinogenic activities depend on 5-HMF content in food products, it is important to the reactive by product SMF (30). In cell culture consider countries with hot climate conditions studies, it has been reported to have weak when defining such limits for 5-HMF content. The genotoxic effects on the HepG2 cell lines (31), 5-HMF limit in honey has been defined as 40 ppm, and it can also cause DNA damage in cells, whereas a level of 80 ppm was considered the irrespective of SULT1A1 activity. However, DNA standard for countries with hot climates (41). In damage caused by 5-HMF has only been observed Turkey, the upper limits for honey, fruit juice and in high concentrations. For instance, a significant molasses products have been defined as 40 level of 5-HMF-induced DNA damage was mg/kg, 20 mg/kg, and 75 mg/kg, respectively reported after exposure to 5-HMF at a (42). concentration of 100 mM for three hours (28). Both 5-HMF and SMF were found to be weak While 5-HMF is formed spontaneously, it is intestinal carcinogens in mice (32). In addition, generally produced during autoclaving. If high concentrations of 5-HMF have been shown to pharmaceutical fluids contain glucose, the heat have irritant effects on the upper respiratory applied during sterilization may cause breakdown airways, eyes, skin and mucosal membranes. and the formation of 5-HMF, and it has been Based on findings obtained from experimental detected in dialysis solutions containing 1 to 60 animals, 5-HMF was suggested to have percent glucose (pH 1-8) that were heat-sterilized tumorigenic and colon cancer-stimulating effects at 121 °C (43). The concentrations detected in (6), and some researchers have reported that 5- sterile glucose solutions, intravenous solutions HMF can act as a neurotoxin, and that its and glucose solutions vary between 1–90 mg/L, accumulation in the body and interactions with 3–56 mg/L and 1–4 mg/L, respectively. 5-HMF proteins may result in muscle and visceral lesions concentrations have also been positively (33). Taking all this into account, it has become a correlated with high acidity (pH<4), high substance of interest for researchers, and the sterilization temperatures (>110 °C) and long presence of 5-HMF in foods has raised sterilization times (30 min) (11). toxicological concerns (29).
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Another study investigated preparations of intravenous injection solutions containing 10 The presence of 5-HMF and similar compounds in percent fructose, which had a pH lower than 3.5– some parenteral preparations containing 4.0 and were sterilized at temperatures between dextrose, solutions prepared for peritoneal 110 and 130 °C. Concentrations of 5-HMF in a dialysis and also some excipients used in newly prepared 50 percent dextrose solution were pharmaceutical sector is a marker of quality for found to be 0.10 μg/mL, while this level increased pharmaceutical products. Based on European to 0.72 μg/mL 24 hours after preparation, and Pharmacopeia, the level of dextrose in solutions following four years of storage at 70 °F (21.1 °C), used for peritoneal dialysis should not exceed 10 5-HMF levels increased to 5.8 μg/mL (44). μg 5-HMF and 25 mg dextrose if they contain no bicarbonate, and 20 μg 5-HMF and 25 mg Fruits contain varying amounts of 5-HMF due to dextrose if they contain bicarbonate (45). While different sugar and organic acid contents (citric, 5-HMF limits are set by the relevant regulations, malic, and ascorbic acids, etc.). Both the highest factors such as technological processes, the sugar and the lowest 5-HMF levels were found in syrup content of medical products, and the storage samples with mixed fruit as flavor agent. Also the conditions of foods and pharmaceutical products second lowest and the second highest levels were should be considered. found in grape flavored syrups. On the other hand, 5-HMF was not detected in syrup samples CONCLUSION containing glycerin as a sweetener. 5-HMF is produced as a result of dehydration of fructose, The storage conditions of medications are glucose, and other reducing sugars and in the generally defined according to mild climatic early stages of the Maillard reaction. Therefore, conditions and low sunlight. If extremely hot 5-HMF formation is not expected in the syrup climatic conditions are not taken into account, the samples containing glycerin. The reason for using stability of medications may become a source of two syrup samples containing glycerin in this significant problems in countries with high study is to contribute to this situation. The results temperature and sunlight levels. None of the show that there is no effect of flavors on 5-HMF syrup samples we analyzed were contain 5-HMF levels. above the legal limits set for foods. Although 5- A positive correlation has been noted in the HMF content was not found to be high in the evaluation of 5-HMF level and expiration date, syrups analyzed in the present study, there may although this cannot be directly associated with still be interactions with the amino groups in the expiration date, as syrup samples contain pharmaceutical formulations, and changes might different types and amounts of sweeteners. There be seen in the activity of such drug products. is also the same situation in food products not Accordingly, even insignificant amounts of 5-HMF only in pharmaceutical formulations. The and similar by products in medical formulations formation of 5-HMF increases in parallel with the and foods should not be ignored. 5-HMF levels storage time of foods. can exceed legal limit in several food items. The only food for which a legal limit on 5-HMF High temperatures induce the formation of 5- concentrations has been set is honey. 5-HMF may HMF. Aside for in one sample, the 5-HMF content exceed tolerable daily intake as a result of taking in syrups kept at 40±2 °C was found to be higher from different sources such as foods and than those kept at room temperature. But this medicine. Therefore monitoring of 5-HMF increase was not statistically significant (p>0.05). contents in foods and medical products used by There may be a significant increase in the longer sensitive populations, especially children seems incubation time at higher temperatures. to be necessary. Syrups containing sweeteners However, our aim was to show that the level could were used in our study. This type of syrups are be increased even in short-term small mostly used by children. Other 5-HMF sources temperature increases. include sugar-containing foods such as honey, jam, fruit juices, which children consume The highest levels were found in two samples that frequently. A low concentration of 5-HMF does not sodium saccharin used as a sweetener. 5-HMF mean that it does not cause toxicity. Because it commonly occurs by the dehydration of can accumulate during both nutrition and medical monosaccharides and disaccharides. A study treatment. investigated the effects of different temperatures and pH values on 5-HMF formation while REFERENCES preparing simple syrups, and based on the findings, the authors concluded that pH and 1. National Center for Biotechnology storage temperature had significant effects on 5- Information. PubChem Compound Database; HMF formation. Accordingly, the best way to CID=237332, avoid 5-HMF formation is to prepare the syrup https://pubchem.ncbi.nlm.nih.gov/compound/23 under high pH conditions while maintaining low 7332 (accessed July 3, 2018). storage temperatures, although there have been no studies to date concerning the effects of pH 2. Zakrzewska ME, Bogel-Lukasik E. 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